Anti Thy 1.2 monoclonal antibody-ricin hybrid utilized as a tumor suppressant

ABSTRACT

A tumor suppressive composition active against lymphoma consisting of an injection of hybrid protein anti Thy 1.2 monoclonal antibody-ricin and hyperosmotic lactose. The inoculation i.v. of murine tissues in vivo by lymphoma is made at -20 to -25 days and the tumor suppressant composition is used i.v. at Day 1 in an amount of 1-3 μg of anti Thy monoclonal antibody-ricin together with sufficient hyperosmotic lactose to raise the lactose level to 20-30 mM. The broad purpose of this invention is to modify the receptor specificity of a potent toxin such as ricin by coupling it with a monoclonal antibody directed at a specific tumor or differentiation antigen. The object here is to use the reagent to selectively kill tumor cells without affecting normal cells.

This invention relates to a tumor suppressive composition active againstlymphoma consisting of an injection of hybrid protein anti Thy 1.2monoclonal antibody-ricin and hyperosmotic lactose. The inoculation i.v.of murine tissues in vivo by lymphoma is made at -20 to -25 days and thetumor suppressant composition is used i.v. at Day 1 in an amount of 1-3μg of anti Thy monoclonal antibody-ricin together with sufficienthyperosmotic lactose to raise the lactose level to 20-30 mM. The broadpurpose of this invention is to modify the receptor specificity of apotent toxin such as ricin by coupling it with a monoclonal antibodydirected at a specific tumor or differentiation antigen. The object hereis to use the reagent to selectively kill tumor cells without affectingnormal cells.

The purpose of this invention is to modify the receptor specificity of apotent toxin such as ricin by coupling it with a monoclonal antibodydirected at a specific tumor or differentiation antigen. The objectgenerally is to use this reagent to selectively kill human tumor cellswithout affecting normal cells.

A number of attempts have been made to develop tumor specific cytotoxicreagents by coupling antitumor antibodies to toxins. Early studiesfailed to show large selectivity between tumor cells and normal cellsbecause (1) toxin binding to normal cells via toxin B chain was notblocked and (2) polyclonal antibodies raised in xenogenic animals havebroad specificity and react with normal cells (Science, 169:68-70, 1970;J. Natl. Cancer Inst., 55:473-477, 1975; Nature, 271:752-755, 1978).

However, in order to overcome toxin binding to normal cells via thetoxin B chain, conjugates with antibody and toxin A chains have beenattempted (the toxin A chain is what kills the cell if it can get to theright intracellular compartment). A chain-antibody conjugates showedcell type specificity but low overall toxicity (Biochem. Biophys. Res.Comm., 90:320-326, 1979).

These antibody toxin conjugates use the entire toxin since it is shownthat the toxin B chain contains a necessary entry function in additionto its usual binding function. The toxin ricin is bound to normal cellswith lactose. In animals studies antibody-ricin conjugates are givenintravenously in hyperosmotic lactose, sufficient to raise serum lactoseto 20-30 mM. The entry function on the toxin B chain is at anintracellular site not accessible to lactose. Therefore, the entryfunction is maintained and the antibody toxin conjugate in the presenceof lactose has the same toxicity as ricin alone toward the target cell.To insure a high degree of tumor specific selectivity, antibodies are ofmonoclonal origin.

USE STATEMENT

This invention is a test or kit with some human useful linkage whichutilizes an antibody toxin conjugate where the entire toxin is utilizedand it is shown that the toxin B chain contains a necessary entryfunction in addition to its usual binding function. In animal studiesantibody ricin conjugates are given intravenously in hyperosmoticlactose sufficient to raise the serum lactose to 20-30 mM.

PRIOR ART STATEMENT

Youle et al, Proc. Natl. Acad. Sci., 76(11):5559-5562, November 1979.

Youle et al, "Anti-Thy 1.2 Monoclonal Antibody Linked to Ricin is aPotent Cell Type Specific Toxin," in press, Proc. Natl. Acad. Sci.

Kohler et al, Nature, 256:495-497, Aug. 7, 1975.

Masuho et al, Biochem. Biophys. Res. Comm., 90(1):320-326, Sept. 12,1979.

Moolten et al, Science, 169:68-70, 1970.

Thorpe, et al, Nature, 271:752-754, February 1978.

It can be shown in Example 4, post, that potent cell type specifictoxins can be made by the proper alteration of toxin bindingspecificity. A monoclonal antibody can be used as the receptor bindingmoiety of a hybrid toxin and will define the hybrid cell typespecificity. By coupling Thy 1.2 specific monoclonal antibody to ricinand blocking the ricin galactose binding site with lactose, the receptorspecificity of ricin is altered. Under these conditions the new reagent,anti-Thy 1.2-ricin, is 700 times more toxic to T-cells which contain theThy 1.2 surface antigen than to cells lacking it. This degree ofselectivity can allow one to kill antigen bearing cells and not othercells in tissue culture. This differential of toxicity may be sufficientto selectively kill antigen bearing cells in vivo.

Several studies of toxins or their subunits linked to polyclonalantibodies, hormones, and lectins have been reported. These hybrids,though generally showing altered receptor specificity of the toxins,have very low potency compared to the native toxins. The M6P-ricin andanti-Thy 1.2-ricin hybrids prepared in this invention maintain the highpotency of the toxin. The mild and specific coupling methods of thisinvention play a role in maintaining high toxin potency. More importantperhaps is the inclusion of the ricin B chain within the hybrid. Thericin B chain performs an entry function which is independent ofordinary ricin binding to surface galactose receptors (Youle et al,Proc. Natl. Acad. Sci., 76:5559-5562, 1979). If a wide variety of highpotency monoclonal antibody hybrids can be made irrespective of theantigen receptor specificity, the ricin B chain entry function becomesmore important. Alternatively, it is possible that the high degree ofselective cytotoxicity elicited by Thy 1.2-ricin is dependent on uniquefeatures inherent in the Thy 1.2 antigen.

Since the M6P-ricin and anti-Thy 1.2-ricin hybrids contain the ricin Bchain, these hybrids require the presence of lactose to block the ricinB chain binding to achieve cell type specificity. This limits thepresently achievable selectivity between cell types to between 30- and700-fold. Naturally occurring toxins which utilize receptor mediatedprotein transport systems can exhibit cell type selectivities up to10,000-fold. This degree of selectivity could in principle be alsoreached by antibody-toxin hybrids of the proper construction. Thecurrently available selectivity, however, is more than ample for the useof hybrids as selective agents for the isolation of receptor minusmutant cell lines. It may be possible to efficiently select for variantsthat lack any cell surface components toward which an antibody can beraised. This approach avoids utilization of complement dependent celllysis.

The use of monoclonal antibodies as the cell recognition moiety of toxinhybrids greatly expands the possible uses of antibody-toxin hybrids.Several cell-type specific and tumor specific or tumor associatedmonoclonal antibodies have been produced. Hybrids of ricin with theseantibodies would kill the antigen bearing cells selectively. There isconsiderable scientific and pharmacologic potential for these potentmonoclonal antibody-ricin hybrids as cell type and tumor specifictoxins.

GENERALIZED TEACHING OF PROCESS

There was previously reported the construction of a cytotoxic reagentspecific for murine T cells carrying the differentiation antigen Thy 1.2and tumor lines derived from such T cells. This reagent was constructedby linking the toxin ricin to a monoclonal antibody directed against theThy 1.2 antigen. This procedure provided the toxin with a new receptorbinding activity specific for the Thy 1.2 antigen. Ricin binding andtoxicity by its usual uptake mechanism was inhibited by blocking thericin galactose site with lactose. In tissue culture anti-Thy 1.2-ricinselectively killed EL-4 leukemia cells carrying the Thy 1.2 antigenwhile failing to kill AKR cells which lacked the Thy 1.2 antigen. Inorder to produce equivalent killing of AKR cells, a 700-fold increase onanti-Thy 1.2-ricin was required.

The present invention shows that the selective cell killing of anti-Thy1.2-ricin in the presence of lactose can be utilized to prolong thelives of B6 mice carrying EL-4 syngeneic tumors. Lactose is supplied tothe extracellular fluid space simultaneously with anti-Thy 1.2-ricin byintravenous injection.

C57BL/6 mice were inoculated with 7×10⁶ EL-4 cells. Twenty-three dayslater mice were selected whose average long axis tumor diameter was 2.5cm. These mice were divided into 5 treatment groups containing 4-6animals each. The average size of the tumors (long×short axis) was4.1±1.5 cm² with a range of 2.5-8.3 cm² and the average size of eachgroup was within the standard error. On Day 23 each treatment group wasinjected via a tail vein with 300 μl of 0.75 M lactose in phosphatebuffered saline and containing either 3, 2, or 1 μg of anti-Thy1.2-ricin, 0.15 μg ricin, or 0.05 μg ricin. Mice were inspected andweighed daily and deaths were tabulated.

MATERIALS AND METHODS

Ricin was purified from seeds of Ricinus communis. L-[U-¹⁴ C]leucine in0.01 N HCl (290 mCi/mmole) was from New England Nuclear, andm-maleimidobenzoyl-N-hydroxysuccinimide ester was from Pierce ChemicalCo. Mouse thymic leukemia cells, EL-4, of B6 background were supplied bythe National Institute of Allergy and Infectious Diseases, AKR-K36thymic leukemia cells by the Fred Hutchinson Research Center, and AKT8,a spontaneous thymoma from AKR mice by Johns Hopkins University.Monoclonal antibody specific for Thy 1.2 mouse T cell antigen, clone30-H12 derived by Ledbetter and Herzenberg (Immunol. Rev.,47:63-90(1979)) was purchased from Becton Dickinson, Mountain View,Calif. The hybridoma was generated by fusion of the mouse myeloma lineNS1 with spleen cells of LOU/Ws1/M rats immunized with SJL (Thy 1.2)mouse spleen cells. This xenogeneic antibody is highly reactive againstmouse cells carrying the Thy 1.2 alloantigen but nonreactive againstthymocytes of AKR mice carrying the Thy 1.1 allele.

EXAMPLE 1

C57BL/6 mice injected subcutaneously with 7×10⁶ EL-4 lymphoma cells 23days earlier were treated by intravenous injection of the hybrid proteinanti-Thy 1.2 monoclonal antibody-ricin and hyperosmotic lactose. Controlanimals received ricin and hyperosmotic lactose. Average tumor weight attreatment was 6 grams. Mean survival times of control groups followingtreatment were 6 and 8 days while groups receiving 2 and 3 μg of hybridwere extended to 13 days. Tumor necrosis was more pronounced in animalstreated with hybrid.

This example demonstrated that monoclonal antibody-ricin hybridsdirected against adult differentiation antigens can be effective tumorsuppressive reagents in vivo. It also demonstrates that lactose which isalso required to achieve selective cytotoxicity with hybrid toxins ofthis type can be administered as a hyperosmotic solution.

EXAMPLE 2

The cell type specificity of the toxin ricin, which ordinarily binds,enters, and kills cells through receptors containing galactose, wasaltered by covalently binding it to a monoclonal antibody and byreversibly binding it to lactose. The antibody, a monoclonal ratIgG_(2b) directed against the Thy 1.2 antigen, provided ricin with a newbinding site for the murine thymus cell surface. The addition of lactosesaturated the galactose binding site on ricin and inhibited ricin frombinding and killing cells via the galactose-containing receptors.

The antibody-ricin hybrid protein, anti-Thy 1.2-ricin, formed with athioether linkage was purified by size exclusion and affinitychromatography. When assayed by inhibition of protein synthesis of EL-4cells which express the Thy 1.2 antigen, anti-Thy 1.2-ricin was equallyas toxic as ricin on a molar basis. The hybrid protein toxicity wasunchanged in the presence of 100 mM lactose, whereas unmodified ricintoxicity was reduced more than 100-fold. This demonstrated the alteredreceptor specificity of the ricin hybrid.

The cell type specificity of the anti-Thy 1.2-ricin inhibition ofprotein synthesis correlated with the presence of the Thy 1.2 antigen.Anti-Thy 1.2-ricin at 4 μg/ml in the presence of lactose inhibitedprotein synthesis within 3.5 hours 60-80% in EL-4 cells but did notaffect protein synthesis in AKR T cell lines from mice which carried theThy 1.1 alloantigen and HeLa cells which lacked the Thy 1 antigen.

Anti-Thy 1.2-ricin in the presence of lactose selectively killed EL-4cells at concentrations which did not kill AKR-K36 cells. Thisselectivity expressed as the ratio of Anti-Thy 1.2 ricin concentrationsrequired to kill 40% of both cell types was 700.

Ricin-monoclonal antibody hybrids of this type combined a high degree ofcell type selectivity and toxicity and has pharmacologic utility asanti-tumor reagents.

EXAMPLE 3

In the successful modification of ricin's native receptor and cell-typespecificity to that dictated by a covalently bound monoclonal antibody,monoclonal anti-Thy 1.2 rat IgG_(2b) was covalently coupled to ricin.This antibody bound to the murine T cell surface differentiation antigenwhich occurred in most strains with the exception of AKR and A strains.When the hybrid anti-Thy 1.2-ricin was exposed to cells in the presenceof lactose, the observed toxicity exhibited cell type specificity of theantibody. Thus, anti-Thy 1.2-ricin plus lactose inhibited proteinsynthesis in murine T cells carrying Thy 1.2 but did not affect murine Tcells of AKR background or a human cell line. Cells carrying Thy 1.2were sensitive to a 700-fold lower concentration of Thy 1.2-ricin pluslactose than cells lacking Thy 1.2 as judged by cell killing.

EXAMPLE 4 Hybrid Synthesis

The bifunctional cross-linking agent,m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) was used to linkricin to IgG similar to the method used to link ricin to diphtheriatoxin A chain as described by Youle et al, J. Biol. Chem.,254:11089-11096, 1979. Ricin, 4 mg protein in 400 μl of 0.01 M Na₂ HPO₄,pH 7.5 was mixed while blending on a vortex miser with 13 μl ofdimethylformamide containing 0.032 mg of MBS giving a molar ratio of 1.7MBS/ricin. The mixture was incubated at room temperature for 30 min.

The monoclonal IgG against Thy 1.2 was prepared for cross-linking bypartial reduction of disulfide bonds. IgG, 0.6 mg of 0.6 ml of bufferedsaline (as supplied from Becton Dickinson), was mixed with 30 μl of 1 Mdithiothreitol and incubated at room temperature for 30 min. The IgG in300 μl aliquots was passed over a Sephadex G-25 column (1.4×5 cm) in 6.7mM Na₂ HPO₄ pH 7.4 and 14 mM NaCl and the peak fractions pooled. Thefinal volume of the pooled IgG fractions was about 3 ml containing 0.6mg protein. The ml of partially reduced IgG was mixed with 400 μl ofMBS-ricin and incubated at room temperature for 2 hours. The mole ratioof ricin:IgG was 18:1.

Hybrid Synthesis and Purification

Artificial hybrid conjugates of ricin and monoclonal antibody againstthe Thy 1.2 cell surface component of mouse thymus cells weresynthesized with the bifunctional cross-linking agent,m-maleimidobenzoyl-N-hydroxysuccinimide as described supra. In laterpreparations the reaction between MBS-ricin and IgG was stopped after 2hrs by making the mixture 6.6 mM with N-ethylmaleimide.

The hybrids were separated from unreacted ricin by high pressure liquidchromatography on a 7.5×600 mm G3000 SW TSK gel permeation column (ToyoSoda USA, 200 Park Avenue, New York, N.Y.). The column was run at a 1ml/min flow rate with 100 mM Na₂ HPO₄, pH 7.2. Anti-Thy 1.2 IgG wasfound to elute from the column after 13.4 ml and ricin after 19 ml.Chromatography of the hybrid mixture of partially reduced IgG andMBS-ricin showed a peak at 12.4 ml and another at 19 ml. Ricin elutedlater than expected on the basis of molecular size presumably due tointeraction with the column matrix.

The peak of hybrid protein, between 10 and 14 ml elution volume wascollected from 17 runs and concentrated to 17 ml by dialysis against 60%sucrose in 6.7 mM Na₂ HPO₄, pH 7.4, 150 mM NaCl buffer. The finalconcentration of protein was between 34 and 85 μg/ml.

The hybrid protein was found to the galactose residues of Sepharose-4Band thereby separated from unreacted IgG. The pooled hybrid protein andIgG fraction from the TSK column was applied to a 2 ml Sepharose-4Bcolumn, washed with 6.7 mM Na₂ HPO₄, pH 7.4, 150 mM NaCl and thericin-IgG hybrids eluted in a sharp peak with 100 mM lactose in the samebuffer. The final yield of hybrid protein was about 95 μg of proteincontaining 30% of the initial IgG protein.

Hybrid Activity

The hybrid proteins, ricin and antibody were assayed for inhibition ofprotein synthesis in T-cell leukemic EL-4 cells which contain the Thy1.2 antigen. The antibody alone at 4,000 ng/ml did not inhibit EL-4 cellprotein synthesis. The hybrid toxin on a weight basis was 4-fold lesspotent than ricin and therefore on a mole basis anti-Thy 1.2-ricinequals or exceeds ricin toxicity. Lactose, which competitively blocksricin binding to cells inhibited toxicity of ricin at least 200-fold inEL-4 cells. In contrast, the hybrid was inhibited less than 2-fold bylactose. Thus, in the presence of 100 mM lactose, EL-4 cells were morethan 100-fold more sensitive to the hybrid protein than to ricin.Mixtures of equal amounts of anti-Thy 1.2 antibody and ricin behavedidentically as ricin.

In the context of this invention, pharmacological amount is thepharmacological effective amount and the amount of anti Thy 1.2monoclonal antibody-ricin is 1-3 μg.

We claim:
 1. A tumor suppressive cytotoxic reagent active on murinelymphoma cancer systems which consists of anti Thy 1.2 monoclonalantibody-ricin.
 2. A composition for treatment of murine neoplasmacomprising a pharmacological active amount therefor of the hybridprotein anti Thy 1.2 monoclonal antibody-ricin in a hyperosmotic lactosesolution.
 3. A murine tumor-suppressive reagent composition comprisingan effective amount therefor of the hybrid protein anti Thy 1.2monoclonal antibody-ricin and lactose in a pharmacological carrier.